1. Technical Field of the Invention
The present invention relates to novel identified polynucleotides (or nucleic acids) and polypeptides (or proteins) or salts thereof, as well as peptide fragments thereof; mutants and derivatives of said polynucleotides (or nucleic acids) or said polypeptides (or proteins); to methods for producing such polynucleotides (or nucleic acids) and polypeptides (or proteins) as well as such mutants and derivatives; to agonists and antagonists of said polypeptides (or proteins); to antibodies against said polypeptides (or proteins), especially monoclonal antibodies thereagainst; and to use of said polynucleotides (or nucleic acids), polypeptides (or proteins), mutants, derivatives, agonists and antagonists.
The present invention relates to novel proteins which belong to the papain family and are cysteine proteinase enzymes (the cysteine proteinase enzymes are expected to be involved in various normal cellular processes, including the turnover of intracellular proteins, prohormone activation, and bone remodeling and to play important roles in a variety of pathological conditions such as Alzheimer""s disease, pulmonary emphysema, rheumatoid arthritis, muscular dystrophy, osteoporosis, neurodegenerative disease, and cancer invasion and metastasis), useful for such studies, particularly to novel human cathepsin proteins (or fragments thereof) or salts thereof and to genes encoding the proteins or fragments. More specifically, the present invention relates to novel human cysteine proteinase-type proteins having a papain-like structure at the active site, said cysteine proteinase protein being cloned from human brain cDNA libraries (the instant novel human cysteine proteinase protein is named xe2x80x9cCathepsin L2xe2x80x9d); to DNA comprising a nucleotide sequence coding for said cysteine proteinase protein; to host cells transformed with said DNA; to processes for producing said human cysteine proteinase protein by said transformed cells and to applications of such proteins and nucleic acid fragments.
2. Description of the Related Art
The cysteine proteinases are a family of enzymes involved in many normal cellular processes, including the turnover of intracellular proteins, prohormone activation, and bone remodeling (Berti, P. J., et al., J. Mol. Biol., 246: 273 to 283, 1995). In addition, it has been suggested that these proteolytic enzymes play important roles in a number of pathological conditions such as Alzheimer""s disease, pulmonary emphysema, rheumatoid arthritis, muscular dystrophy, osteoporosis, and cancer invasion and metastasis (Berti, P. J., et al., J. Mol. Biol., 246: 273 to 283, 1995; Berquin, I. M., et al., Perspect. Drug Discov. Des., 2: 371 to 388, 1994).
At present, eight human cysteine proteinases of the papain family have been isolated and characterized at the amino acid sequence level: cathepsin B (Chan, S. J., et al., Proc. Natl. Acad. Sci. U.S.A., 83: 7721-7725, 1986), cathepsin L (Gal, S., et al., Biochem. J., 253: 303-306, 1988), cathepsin H (Ritonja, A., et al., FEBS Lett., 228: 341-345, 1988), cathepsin S (Shi, G. P., et al., J. Biol. Chem., 267: 7258-7262, 1992), cathepsin C (Paris, A., et al., FEBS Lett., 369: 326-330, 1995), cathepsin O (Velasco, G., et al., J. Biol. Chem., 269: 27136-27142, 1994), cathepsin K (Inaoka, T., et al., Biochem. Biophys. Res. Commun., 206: 89-96, 1995) and cathepsin W (Linnevers, C., et al., FEBS Lett., 405: 253-259, 1997).
Furthermore, several groups have described the existence of additional cysteine proteinases including cathepsins S, M, N, P, and T, which were originally identified because of their degrading activity on specific substrates such as aldolase, collagen, proinsulin, or tyrosine aminotransferase, but whose characterization at the molecular level has not yet been reported (Pontremoli, S., et al., Arch. Biochem. Biophys., 214: 376-385, 1982; Maciewicz, R., et al., Biochem. J., 25: 433-440, 1988; Docherty, K., et al., Proc. Natl. Acad. Sci. U.S.A., 79: 4613-4617, 1982; Gohda, E., et al., J. Biol. Chem., 256: 2567-2572, 1981).
Structural comparisons between the different members of the cysteine proteinase family have shown that they are synthesized as preproenzymes, which are processed to the corresponding proenzymes and targeted to the lysosomes by the mannose 6-phosphate signal attached to them. However, in some cases, the precursors of these lysosomal enzymes escape from this processing pathway and continue along the secretory route, entering storage granules and finally being released into the extracellular space (Sloane, B. F., et al., Science, 212: 1151-1153, 1981). Amino acid sequence comparisons between all members of the family have revealed that they are not closely related; the percentage of identity between them is less than 50%. Nevertheless, in their amino acid sequences, all of them contain a series of amino acids that are absolutely conserved and essential for their catalytic activity (Berti, P. J., et al., J. Mol. Biol., 246: 273-283, 1995).
Because it seems clear that cysteine proteinases play essential roles in both normal and pathological conditions, including tumor processes, over the last few years, the possibility that additional, uncharacterized members of this family of proteolytic enzymes could be produced by human tumors has been examined. This search for new human cysteine proteinases led us to identify cathepsin O, which was originally cloned from a breast carcinoma but is widely distributed in human tissues (Velasco, G., et al., J. Biol. Chem., 269: 27136-27142, 1994). Furthermore, the cloning and characterization of human bleomycin hydrolase, a cytosolic cysteine proteinase that is distantly related to other members of the papain family and is involved in chemotherapy resistance, has recently been reported (Ferrando, A. A., et al., Cancer Res., 56: 1746-1750, 1996.).
It would be expected that the cysteine proteinases are not only involved in many normal cellular processes, including the turnover of intracellular proteins, prohormone activation, and bone remodeling but also play important roles in a number of diseases, disorders and pathological conditions such as Alzheimer""s disease, pulmonary emphysema, rheumatoid arthritis, muscular dystrophy, osteoporosis, neuronal degenerative disease and cancer invasion and metastasis. Accordingly, it would be important to identify and isolate a novel cysteine proteinase, followed by elucidating the function of said cysteine proteinase involved in various diseases and disorders, especially cancers in view of not only elucidation of critical mechanism leading to such diseases and disorders but also researches and developments of therapy and therapeutic drugs thereagainst.
The present inventors have taken a view that, in the cysteine proteinase family, there will be novel members which have not been reported yet. The present inventors have carried out various studies by means of genetic engineering techniques. As a result, they have succeeded in cloning a human gene coding for a novel cysteine proteinase member and have disclosed all of its gene nucleotide sequence and amino acid sequence whereupon the present invention has been accomplished. When the putative amino acid sequence of the isolated novel cysteine proteinase was compared with the sequences of the already-reported cathepsins, it was noted that it has 78% homology to cathepsin L and not more than 40% homology to other cathepsins, respectively. From this characteristic feature in the amino acid sequence, said novel member was named xe2x80x9ccathepsin L2xe2x80x9d.
Accordingly, the present invention provides novel proteins having cathepsin L2 activity, of which origin is human, in particular, polypeptides (or proteins) called xe2x80x9ccathepsin L2xe2x80x9d herein, processes for producing the same and use thereof, genes (or polynucleotides or nucleic acids) coding for said proteins, applications thereof, etc.
The present invention provides probes for hybridization, specific to cathepsin L2 genes. Further, the present invention provides antibodies specifically reactive to cathepsin L2, methods for immunologically assaying cathepsin L2 which comprises using said antibody, and assay kits therefor.
The present invention relates to novel human cathepsin L2 genes, to cathepsin L2 proteins which originated from human, and to analogues thereof. The present invention also relates to novel DNA sequences coding for all or part of said human cathepsin L2, to vectors comprising such DNA sequences, and to host cells transformed or transfected with such vectors. Further, the present invention encompasses processes for producing recombinant human cathepsin L2 and uses thereof. The present invention also relates to nucleic acids, such as DNA and RNA probes, hybridizable to human cathepsin L2 genes. The present invention further relates to antibodies, particularly monoclonal antibodies, which bind to human cathepsin L2, and to hybridomas which produce said antibodies. The present invention provides reagents for detecting and/or assaying human cathepsin L2, which comprise said products, and methods for detecting and/or assaying human cathepsin L2, which comprise using said reagents.
The present invention provides human cathepsin L2 agonists. Among them, more desired agonists are cathepsin L2 pseudomolecules which bind with cathepsin L2 binding molecules or cathepsin L2 receptor molecules and/or either produce or enhance cathepsin L2 inductive actions, and the like. Preferred agonists are also molecules which interact with cathepsin L2, cathepsin L2 polypeptides, or other regulating substances for cathepsin L2 activity to either enable or strengthen cathepsin L2 actions and effects or more.
The present invention provides human cathepsin L2 antagonists. Among them, more desired antagonists are cathepsin L2 pseudomolecules which bind with cathepsin L2 binding molecules or cathepsin L2 receptor molecules but do not produce one or more cathepsin L2 inductive actions, or which inhibit such actions, and the like. Preferred antagonists are also molecules which interact with cathepsin L2, cathepsin L2 polypeptides, or other regulating substances for cathepsin L2 activity to either inhibit or suppress one or more cathepsin L2 actions and effects.
The present invention further provides methods for detecting not only substances which inhibit a cysteine proteinase activity associated with human cathepsin L2 but also substances which inhibit a proteolytic activity associated therewith. The present invention thus relates to drugs comprising such substances or derivatives thereof as effective components, for example, neutralizing antibodies which inhibit its cysteine proteinase activity or proteolytic activity, and specifically to monoclonal antibodies which bind with human cathepsin L2. Further, the present invention relates to peptide fragments of human cathepsin L2 and derivatives thereof or a salt thereof, which bind with anti-human cathepsin L2 antibodies but have no human cathepsin L2 activity. The present invention relates to antisense nucleotide and derivatives thereof which inhibits human cathepsin L2 expression, and to agents which comprise the same as an active component.
The present invention provides:
(1) a novel protein or a salt thereof, which
(A) belongs to a member of cathepsins derived from human; and
(B) is (i) human cathepsin L2 protein or (ii) any of those having at least 80% homology to an amino acid sequence of said human cathepsin L2 protein and possessing (a) a cysteine proteinase activity or (b) an antigenicity substantially equivalent to said human cathepsin L2 protein;
(2) a protein or a salt thereof, which has an amino acid sequence selected from the group consisting of
(a) amino acid residues 114 to 334,
(b) amino acid residues 18 to 334, and
(c) amino acid residues 1 to 334, of SEQ ID NO: 1 in the Sequence Listing, and
(d) substantially equivalent amino acid sequences thereto;
(3) a peptide fragment of the protein according to above (1) or (2), or a salt thereof;
(4) a nucleic acid comprising a nucleotide sequence coding for the protein according to above (1) or (2);
(5) the nucleic acid according to above (4), having (i) an open reading frame region of the nucleotide sequence of SEQ ID NO: 2 in the Sequence Listing or (ii) a substantially equivalent thereto;
(6) a vector comprising the nucleic acid according to above (4) or (5);
(7) a transformant or transfectant harboring (i) the nucleic acid according to above (4) or (5) or (ii) the vector according to above (6);
(8) the transformant or transfectant according to above (7), wherein a host cell for the nucleic acid or vector is selected from the group consisting of Escherichia coli, yeast, CHO cell, and COS cell;
(9) the protein according to above (1) or (2), which is a product obtained from the transformant or transfectant according to above (8);
(10) a probe for hybridization to human cathepsin L2 gene, comprising all or part of the nucleotide sequence of SEQ ID NO: 2;
(11) the probe according to above (10), comprising a untranslation region of the nucleotide sequence of SEQ ID NO: 2;
(12) an antibody against
(i) the protein or a salt thereof according to above (1);
(ii) the protein or a salt thereof according to above (2); or
(iii) the peptide fragment or a salt thereof according to above (3);
(13) an immunological assay for detecting and/or measuring the protein or a salt thereof according to above (1) or (2), which comprises using, as a reagent, a monoclonal antibody specifically immunoreactive with the protein or a salt thereof according to above (1) or (2);
(14) an immunological assay agent for detecting and/or measuring the protein or a salt thereof according to above (1) or (2), which comprises a monoclonal antibody specifically immunoreactive with the protein or a salt thereof according to above (1) or (2);
(15) a pharmaceutical composition comprising (i) the protein or peptide fragment or a salt thereof according to any of above (1) to (3); (ii) the nucleic acid according to above (4) or (5); or (iii) the antibody according to above (12);
(16) a pharmaceutical composition comprising a compound which enhances or inhibits a biological activity of the protein or peptide fragment or a salt thereof according to any of above (1) to (3), or a salt thereof; and
(17) a screening method or screening kit for a compound which enhances or inhibits the biological activity of the protein or peptide fragment or a salt thereof according to any of above (1) to (3), or a salt thereof.
In another aspect, the present invention provides:
(18) the protein or a salt thereof according to above (1), wherein the protein (1) is native human cathepsin L2 or a salt thereof, or (2) has a cysteine proteinase activity or antigenicity, or primary structural conformation identical with or substantially equivalent to that of native human cathepsin L2, or a segment thereof;
(19) the protein or a salt thereof according to above (1), wherein the protein is at least 80% homologous, preferably 85% or more homologous, or more preferably 90% or more homologous, to an amino acid sequence selected from the group consisting of
(a) amino acid residues 114 to 334,
(b) amino acid residues 18 to 334, and
(c) amino acid residues 1 to 334, of SEQ ID NO: 1 in the Sequence Listing, or a segment thereof;
(20) the protein or a salt thereof according to above (1), wherein the protein is
(i) a human cathepsin L2 protein having an amino acid sequence selected from the group consisting of
(a) amino acid residues 114 to 334,
(b) amino acid residues 18 to 334, and
(c) amino acid residues 1 to 334, of SEQ ID NO: 1 in the Sequence Listing, or
(ii) a member selected from the group consisting of
(d) deletion analogues wherein one or more amino acid residues (for example, 1 to 73 residues, preferably 1 to 60 residues, more preferably 1 to 40 residues, further more preferably 1 to 20 residues, particularly 1 to 10 residues, etc.) characteristic of human cathepsin L2 are deleted,
(e) substitution analogues wherein one or more amino acid residues (for example, 1 to 73 residues, preferably 1 to 60 residues, more preferably 1 to 40 residues, further more preferably 1 to 20 residues, particularly 1 to 10 residues, etc.) characteristic of human cathepsin L2 are replaced with other amino acid residues, and
(f) addition analogues wherein one or more amino acid residues (for example, 1 to 80 residues, preferably 1 to 60 residues, more preferably 1 to 40 residues, further more preferably 1 to 20 residues, particularly 1 to 10 residues, etc.) are inserted, or a segment thereof;
(21) a nucleic acid comprising a nucleotide sequence coding for the protein according to any of above (18) to (20);
(22) a nucleic acid comprising a region selected from the group consisting of
(a) sequences having at least 79% homology to human cDNA of GENBANK(trademark)/EMBL accession number: Y14734,
(b) sequences having at least 80% homology to human cDNA of GENBANK(trademark)/EMBL accession number: Y14734,
(c) sequences having at least 85% homology to human cDNA of GENBANK(trademark)/EMBL accession number: Y14734,
(d) sequences having at least 90% homology to human cDNA of GENBANK(trademark)/EMBL accession number: Y14734,
(e) sequences having at least 95% homology to human cDNA of GENBANK(trademark)/EMBL accession number: Y14734,
(f) sequences having at least 97% homology to human cDNA of GENBANK(trademark)/EMBL accession number: Y14734,
(g) sequences having at least 98% homology to human cDNA of GENBANK(trademark)/EMBL accession number: Y14734, and
(h) sequences having at least 99% homology to human cDNA of GENBANK(trademark)/EMBL accession number: Y14734;
(23) a nucleic acid comprising a region selected from the group consisting of
(a) sequences having at least 79% homology to human cDNA of microorganism deposit NIBH accession No. FERM BP-6640, or human cDNA in recombinant plasmid pGEX-3X CathL2 harbored in microorganism deposit NIBH accession No. FERM BP-6641,
(b) sequences having at least 80% homology to said human cDNA,
(c) sequences having at least 85% homology to said human cDNA,
(d) sequences having at least 90% homology to said human cDNA,
(e) sequences having at least 95% homology to said human cDNA,
(f) sequences having at least 97% homology to said human cDNA,
(g) sequences having at least 98% homology to said human cDNA, and
(h) sequences having at least 99% homology to said human cDNA;
(24) a nucleic acid comprising a region selected from the group consisting of
(a) sequences having at least 79% homology to the nucleotide sequence of SEQ ID NO: 2 in the Sequence Listing,
(b) sequences having at least 80% homology to the nucleotide sequence of SEQ ID NO: 2,
(c) sequences having at least 85% homology to the nucleotide sequence of SEQ ID NO: 2,
(d) sequences having at least 90% homology to the nucleotide sequence of SEQ ID NO: 2,
(e) sequences having at least 95% homology to the nucleotide sequence of SEQ ID NO: 2,
(f) sequences having at least 97% homology to the nucleotide sequence of SEQ ID NO: 2,
(g) sequences having at least 98% homology to the nucleotide sequence of SEQ ID NO: 2, and
(h) sequences having at least 99% homology to the nucleotide sequence of SEQ ID NO: 2;
(25) a vector comprising the nucleic acid according to any of above (21) to (24);
(26) a transformant or transfectant harboring (i) the nucleic acid according to any of above (21) to (24) or (ii) the vector according to above (25);
(27) the transformant or transfectant according to above (26), wherein a host cell for the nucleic acid or vector is selected from the group consisting of Escherichia coli, yeast, CHO cell, and COS cell;
(28) the protein according to any of above (18) to (20), which is a product obtained from the transformant or transfectant according to above (27);
(29) a labeled or non-labeled probe for hybridization, comprising a nucleotide sequence hybridizable with a human cathepsin L2 gene but not hybridizing to a human cathepsin L gene, under high-stringency conditions, wherein the nucleotide sequence has at least part of the nucleotide sequence of SEQ ID NO: 2 in the Sequence Listing;
(30) a method for assaying a nucleic acid encoding human cathepsin L2 in a sample, which comprises the steps of
(i) hybridizing a probe specific to the human cathepsin L2-coding nucleic acid with the sample under conditions effective for specific hybridization with said nucleic acid to form a hybrid, and
(ii) assaying the hybridization of said probe with the sample nucleic acid,
wherein said probe comprises a segment with 10 or more base pairs, said segment which is at least 79% homologous to the nucleotide sequence of SEQ ID NO: 2 in the Sequence Listing;
(31) the antibody according to above (12), which is a monoclonal antibody immobilized on a solid phase;
(32) the antibody according to above (12), which is a labeled monoclonal antibody;
(33) the antibody according to above (12), which is labeled with an enzyme;
(34) the antibody according to above (12), which is labeled Fab"" derived from a monoclonal antibody;
(35) a method for inhibiting the biological activity (e.g., cysteine proteinase activity, antigenicity, etc.) of the protein according to above (1) or (2); and
(36) the method according to above (35), wherein the inhibitor is selected from the group consisting of peptides, proteins, nonpeptide compounds, synthetic compounds, fermented products, cell extracts, plant extracts, animal tissue extracts, and antibodies such as monoclonal antibodies.
In yet another aspect, the present invention provides:
(37) a screening method for a compound capable of enhancing or inhibiting the biological activity of the protein according to any of above (1) to (3) and (18) to (20), a peptide fragment thereof, or a salt thereof, which comprises using, as a tool, a member selected from the group consisting of the proteins according to any of above (1) to (3) and (18) to (20), peptide fragments thereof, and salts thereof;
(38) a screening method for a compound which enhances or inhibits the biological activity of the protein according to any of above (1) to (3) and (18) to (20), a peptide fragment thereof, or a salt thereof, which comprises comparing (i) a first measurement wherein a substrate is contacted with a member selected from the group consisting of the proteins according to any of above (1) to (3) and (18) to (20), peptide fragments thereof, and salts thereof, with (ii) a second measurement wherein the substrate and a test compound are contacted with said member;
(39) a screening kit for a compound capable of enhancing or inhibiting the biological activity of the protein according to any of above (1) to (3) and (18) to (20), a peptide fragment thereof, or a salt thereof, which comprises a member selected from the group consisting of the proteins according to any of above (1) to (3) and (18) to (20), peptide fragments thereof, and salts thereof;
(40) a compound or a salt thereof, capable of enhancing or inhibiting the biological activity of the protein according to any of above (1) to (3) and (18) to (20), a peptide fragment thereof, or a salt thereof, which is obtained or identified (i) by the screening method according to above (17), (37) or (38) or (ii) via using the screening kit according to above (17) or (39); and
(41) a pharmaceutical composition comprising an effective amount of a compound or a salt thereof which enhances or inhibits the biological activity of the protein according to any of above (1) to (3) and (18) to (20), a peptide fragment thereof, or a salt thereof, said compound being obtained or identified (i) by the screening method according to above (17), (37) or (38) or (ii) via using the screening kit according to above (17) or (39).
In a further aspect, the present invention provides:
(42) an immunological assay for the protein or a salt thereof according to above (1), which comprises using, as an assay agent, a monoclonal antibody which specifically immunoreacts with the protein or a salt thereof according to above (1);
(43) an immunological assay agent for the protein or a salt thereof according to above (1), which comprises a monoclonal antibody which specifically immunoreacts with the protein or a salt thereof according to above (1);
(44) the assay method according to above (13), wherein the antibodies according to above (12) are employed, said antibodies comprising at least 2 members composed of both (i) an antibody immobilized on a solid phase and (ii) a labeled antibody;
(45) the assay method according to above (44), wherein the protein or a salt thereof according to above (1) is quantitatively measured;
(46) the assay method according to above (44) or (45), wherein a sample or specimen to be assayed is selected from the group consisting of blood, serum, plasma, articular fluid, urine, saliva, cerebrospinal fluid, amniotic fluid, tissue extracts, tissue culture extracts, cell culture supernatants;
(47) an assay reagent set for detecting and/or assaying human cathepsin L2, which comprises at least 2 members composed of both (i) a reagent comprising an antibody immobilized on a solid phase and (ii) a reagent comprising a labeled antibody, said antibodies each being selected from the antibodies according to above (12) and said assay reagent set being used in the immunological assay according to any of above (44) to (46);
(48) a hybridoma producing a monoclonal antibody against a member selected from the group consisting of (a) the proteins according to above (1) and salts thereof, and (b) peptide fragments thereof and salts thereof, said hybridoma being generated by cell fusion of (i) a spleen cell obtained from an animal, such as a mouse, immunized with said member with (ii) an immortal cell, such as a myeloma cell, derived from an animal, such as a mouse; and
(49) a process for producing the hybridoma according to above (48), which comprises:
fusing a spleen cell obtained from an animal, such as a mouse, immunized with a member selected from the group consisting of (a) the proteins according to above (1) and salts thereof, and (b) peptide fragments thereof and salts thereof, with an immortal cell, such as a myeloma cell, derived from an animal such as a mouse, and
selecting a hybridoma cell capable of producing a monoclonal antibody against said member;
In a still further aspect, the present invention provides:
(50) a nucleic acid containing a nucleotide sequence hybridizing with the nucleotide sequence of SEQ ID NO: 2 under stringent conditions;
(51) a vector containing the nucleic acid according to above (50);
(52) a transformant or transfectant comprising the vector according to above (51);
(53) a protein or a salt thereof, which is a product generated as a consequence of gene expression by the transformant or transfectant according to above (52);
(54) the nucleic acid according to any of above (4), (5), (21) to (24) and (50), wherein the nucleic acid is DNA.
(55) a process for preparation of a product selected from the group consisting of (a) the proteins according to above (1) and salts thereof, and (b) peptide fragments thereof and salts thereof, which comprises:
culturing the transformant or transfectant according to above (7), (8), (26), (27) or (52) to produce and accumulate said product, and collecting or isolating said product;
(56) a compound or a salt thereof enhancing the biological activity of the protein according to any of above (1) to (3), a peptide fragment thereof or a salt thereof, which is a product obtained or identified as a consequence of application of (i) the screening method according to any of above (17), (37) or (38) or (ii) the screening kit according to above (17) or (39);
(57) a compound or a salt thereof inhibiting the biological activity of the protein according to any of above (1) to (3), a peptide fragment thereof or a salt thereof, which is a product obtained or identified as a consequence of application of (i) the screening method according to any of above (17), (37) or (38) or (ii) the screening kit according to above (17) or (39);
(58) the screening method according to above (17), wherein the biological activity of above (17) is of proteinase activity;
(59) the screening kit according to above (17), wherein the biological activity of above (17) is of proteinase activity;
(60) the compound or a salt thereof according to above (40), wherein the biological activity of above (40) is of proteinase activity;
(61) the pharmaceutical composition according to above (16), wherein the biological activity of above (16) is of proteinase activity;
(62) a method for quantitative assay of the protein according to any of above (1) to (3), a peptide fragment thereof or a salt thereof in a sample to be assayed, which comprises:
reacting competitively the antibody according to above (12) with the sample in the presence of a labeled protein according to any of above (1) to (3), a labeled peptide fragment thereof or a salt thereof, and
measuring the amount rate of said labeled protein (or said labeled peptide fragment) or a salt thereof, bound to said antibody; and
(63) a method for quantitative assay of the protein according to any of above (1) to (3), a peptide fragment thereof or a salt thereof in a sample to be assayed, which comprises:
reacting an antibody immobilized on a solid phase according to above (12) with the sample in the presence of a labeled protein according to any of above (1) to (3), a labeled peptide fragment thereof or a salt thereof, and
measuring either the amount of labels immobilized on said solid phase or the amount of labels not binding to said solid phase.
The above objects and other objects, features, advantages, and aspects of the present invention are readily apparent to those skilled in the art from the following detailed description of preferred embodiments. It should be understood, however, that the description of the specification including the following detailed description of preferred embodiments, examples, etc. is illustrating preferred embodiments of the present invention and given only for explanation thereof. It will become apparent to the skilled in the art that a great number of variations and/or alterations (or modifications) of this invention may be made based on knowledge from the disclosure in the following parts and other parts of the specification without departing from the spirit and scope thereof as disclosed herein. All of the patent publications and reference documents cited herein for illustrative purposes are hereby incorporated by reference into the present disclosure.
The term xe2x80x9cand/orxe2x80x9d used herein means the presence of both (1) a jointly connecting relation and (2) a selectively connecting relation. For example, in the case of xe2x80x9ctherapeutic and/or prophylacticxe2x80x9d, it is used in such a sense that said expression covers both (1) xe2x80x9ctherapeutic and prophylacticxe2x80x9d and (2) xe2x80x9ctherapeutic or prophylacticxe2x80x9d. In other cases, the term xe2x80x9cand/orxe2x80x9d is used in the same sense that it covers both (1) a jointly connecting relation and (2) a selectively connecting relation as well.